Antibodies are the effector molecules of the humoral immune response in mammals (B. Alberts et al., Molecular Biology of the Cell (Garland Publishing, Inc. 1994); E. Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). Also known as immunoglobulins (Ig), they are produced by B-lymphocytes in response to antigen stimulation. Each B-lymphocyte produces an antibody with a defined specificity for a particular antigen. During an infection, an individual will generally produce multiple unique B-lymphocyte clones, each expressing and secreting a single type of antibody directed at an antigen expressed by the infectious organism. Following the resolution of the infection, the newly-generated B-lymphocytes enter a quiescent state characterized by minimal proliferation and antibody secretion. These quiescent B-lymphocytes can last for the lifetime of the individual and serve as an immunological memory that can be quickly tapped should the individual again encounter the same infectious organism.
The development of monoclonal antibody technology in the 1970s greatly facilitated the study of antibody biology and the adaptation of antibodies for use in research and medicine (B. Alberts et al., Molecular Biology of the Cell (Garland Publishing, Inc. 1994); G. Kohler et al., Nature 256:495 (1975)). Monoclonal antibodies are produced by hybrid cells that result from a fusion between normal B-lymphocytes and myeloma cells. The myeloma cell lines used for fusion are B-lymphocyte tumor cell lines that grow well in vitro and can propagate indefinitely, in contrast to normal B-lymphocytes that cannot replicate or produce antibody in vitro for more than a few days. Cells derived from a fusion of the two types of cells combine the in vitro growth characteristics of the myeloma cell line with the production of an antibody derived from the B-lymphocyte.
Hybrid cells (hybridomas) are generally produced from mass fusions between murine splenocytes, which are highly enriched for B-lymphocytes, and myeloma “fusion partner cells” (B. Alberts et al., Molecular Biology of the Cell (Garland Publishing, Inc. 1994); E. Harlow et al., Antibodies. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). The cells in the fusion are subsequently distributed into pools that can be analyzed for the production of antibodies with the desired specificity. Pools that test positive can be further subdivided until single cell clones are identified that produce antibodies of the desired specificity. Antibodies produced by such clones are called monoclonal antibodies.
Monoclonal antibodies have many advantages that make them particularly useful in research and medicine. They can be produced in large quantities and often have high and specific affinities for their particular antigens. However, their enormous potential utility is counter-balanced by the difficulty in producing antibodies suitable for pharmaceutical use. This is because the current state of the art for monoclonal antibody production is most effective in the production of murine antibodies. Murine antibodies are recognized by the human immune system as foreign. Patients may have allergic or anaphylactic reactions to the antibodies, or may develop their own antibodies directed against the murine antibodies. This can lead to the formation of large immune complexes that can precipitate in tissues and cause serum sickness, a syndrome consisting of fever, muscle and joint aches, rash, and renal and cerebrovascular injury. Consequently, murine antibodies are of limited value for use in humans.
Many investigators have attempted to generate human monoclonal antibodies by generating hybridomas with human B-lymphocytes (N. Chiorazzi et al, J. Exp. Med. 156:930 (1982); C. M. Croce et al., Nature 288:488 (1980); P. A. Edwards et al, Eur. J. Immunol. 12:641 (1982); R. Nowinski et al, Science 210:537 (1980); L. Olsson et al, Proc. Natl. Acad. Sci. USA 77:5429; J. W. Pickering et al, J. Immunol. 129:406 (1982)). Unfortunately, hybrid cells exhibit poor growth in vitro, low levels of antibody expression, instability of antibody expression, and a poor ability to be cloned by limiting dilution. The explanation for these phenotypes has not been elucidated. Accordingly, most investigators have concluded that the production of human monoclonal antibodies through the generation of hybrid cells formed with human B-lymphocytes is not feasible.
Consequently, diverse and cumbersome approaches have been used to produce human monoclonal antibodies. These include “humanizing” mouse antibodies by creating hybrid murine/hybrid immunoglobulin genes and generating antibodies in transgenic mice that bear human immunoglobulin gene loci. However, these methods are only able to produce antibodies that have been generated in mice by the murine immune system. They do not allow the isolation, production, and use of the naturally-occurring antibodies, the immunological memory that the human immune system produces in response to infections and other antigen exposures. The ability to make monoclonal antibodies directly from human B-lymphocytes is therefore needed and would be of considerable value.